By reversibly breaking and resealing DNA, the topoisomerase I of eukaryotic cells assists in the orderly readout and replication of the genetic information. However, under conditions in which the resealing step of topoisomerase cycle fails, the covalent intermediate in which the enzyme is joined to DNA can become a lethal strand interruption. We have discovered that eukaryotes contain a ubiquitous activity that specifically hydrolyzes the bond connecting topoisomerase I to DNA. To test the hypothesis that this activity functions in the repair of topoisomerase I dead-end covalent complexes, we are carrying out a genetic and biochemical characterization of the enzyme found in the budding yeast, S. cerevisiae. Our first goal is to clone the yeast gene. Toward this end we purified the yeast enzyme far beyond that described in our initial publication and obtained amino acid sequence information from the three polypeptide species in our most highly purified preparation. Two of these proved to be tRNA synthetases and the third a pseudo-uridine synthetase. Since none of these enzymes are suspected to have phosphodiesterase activity they are unlikely candidates to encode the tyrosine-DNA phosphodiesterase in the preparation. Nevertheless, we over expressed tRNA synthetase and knocked out the yeast gene encoding pseudo-uridine synthetase; expression of tyrosine DNA phosphodiesterase was unaffected. We conclude that the enzyme must have a high turnover number and is therefore a minor component of our most highly purified preparation. Rather than pursue reverse genetics by scaling up the enzyme purification from its already substantial scale, we have turned to the study of a genetic variant of S. cerevisiae that has low enzyme activity. In previous work we isolated this variant by brute force screening of heavily mutagenized cells. To study the effect of this mutation in isolation, we have outcrossed it repeatedly to the parental line from which it was isolated. In each of five such rounds of outcrossing, the enzymatic defect has behaved as if it were caused by a single mutation. But, many other characteristics of the initial strain, including sensitivity to growth in the presence of the topoisomerase poison campthothecin, are removed by outcrossing. The lack of the effect of the mutation on sensitivity to campthothecin shows that enzymatic activity is not the only pathway by which yeast can repair topoisomerase damage. To sensitize the cell to effects of such poisons, we had introduced the rad9 mutation so as to eliminate the capacity of yeast cells to undergo check point arrest while other, possibly minor, pathways repair campthothecin induced damage. Cells that bear a rad9 mutation as well as one that leads to lower tyrosine-DNA phosphodiesterase activity are at least five times more sensitive to the lethal affects of campthothecin than are rad9 control cells. This result provides the first evidence that connects tyrosine-DNA phosphodiesterase activity with repair of topoisomerase damage and provides a phenotype that can be used to clone the affected gene.